Construction and characterization of a Borrelia burgdorferi strain with conditional expression of the essential telomere resolvase, ResT

Abstract

Borrelia species are unique in the bacterial world in possessing segmented genomes which sometimes contain over 20 genetic elements. Most elements are linear and contain covalently closed hairpin ends requiring a specialized process, telomere resolution, for their generation. Hairpin telomere resolution is mediated by the telomere resolvase, ResT. Although the process has been studied extensively in vitro, the essential nature of the resT gene has precluded biological studies to further probe the role of ResT. In this work, we have generated a B. burgdorferi strain that carries an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible resT gene controlled by a tightly regulated promoter. ResT is expressed in this strain at ~14,000 monomers per cell, similar to the ~15,000 monomers observed for the parental strain. We demonstrate ResT depletion with a half-life of 16 h upon IPTG washout. ResT depletion resulted in arrested growth 48 h after washout. Interestingly, not all spirochetes died after ResT washout, and at least 15% remained quiescent and could be resuscitated even at 2 weeks postwashout. Significant levels of DNA synthesis were not observed upon growth arrest, suggesting that ResT might interact directly or indirectly with factors controlling the initiation or elongation of DNA synthesis. Analysis of the linear plasmids lp17 and lp28-2 showed that the linear forms of these plasmids began to disappear and be replaced by higher-molecular-weight forms by 24 h post-IPTG washout. Treatment of DNA from the ResT-depleted strain with ResT in vitro revealed the presence of replicated telomeres expected in replication intermediates.

FIG 1

Replication pathway for linear replicons in Borrelia species. Arrows indicate hairpin telomeres at the left (L) and right (R) ends. Initiation occurs at the central origin (13), and complete replication results in the formation of a head-to-head (L′L)–tail-to-tail (R-R′) dimer. The lines bisecting the junctions denote axes of 180° rotational symmetry. Where in the cell telomere resolution occurs is unknown, as is whether telomere resolution is concerted at both ends or whether it occurs independently at each end. This figure is adapted from reference with permission.

FIG 2

ResT half-life in B. burgdorferi. (A) To measure the half-life of ResT protein in the conditional expression strain (GCB2127), a time course analysis was performed using Western blotting following IPTG washout. B. burgdorferi was grown for 48 h in the presence of 1 mM IPTG until it reached late exponential phase. Following IPTG washout, the culture was diluted to 1.0 × 10⁶ spirochetes/ml (time zero) and incubation was continued for 48 additional hours, with aliquots removed at the indicated times. For Western blotting, 2.0 × 10⁷ spirochetes were loaded per well as described in Materials and Methods. ResT from the wild-type parent, B31-A, is also shown. Band intensities were quantified and analyzed using AlphaImager software (Alpha Innotech). (B) The half-life of ResT was calculated by plotting the percentage of ResT remaining versus time. Data in the graph represent two separate experiments. The calculated ResT half-life was 16 h.

FIG 3

(A) Growth curves of the resT knockout mutant GCB2127. B. burgdorferi cultures were grown in BSK II medium with and without IPTG at 35°C, and spirochetes were counted every 24 h. After 48 h, the cultures were diluted with an equal volume of medium to avoid entry into stationary phase and were counted before and after dilution at the 48-h time point. Growth of the wild type, B31-A (GCB908), was compared to growth of the resT-inducible strain (GCB2127). (B) Spirochetes from cultures at the indicated times following IPTG washout were stained with the LIVE/DEAD BacLight staining kit (Invitrogen) to determine if cells were alive or dead. Spirochetes were analyzed by fluorescence microscopy for staining with Syto-9 (live) or propidium iodide (dead). Ten fields of view were analyzed for each time point. Living spirochetes were also enumerated by dilution in duplicate of the cultures and plating in BSK II medium plus IPTG in 96-well microtiter plates. The percent living cells for counting was based upon the total number of spirochetes counted by dark-field microscopy. At each time point, the mean percentage of living cells ± the standard error is plotted for both the LIVE/DEAD staining and plating methods.

FIG 4

Determination of the average spirochete length of the wild-type (GCB908) and resT-inducible (GCB2127) strains. The length of individual DAPI-stained spirochetes was determined at the indicated times after IPTG washout using ImageJ (NIH) as described in Materials and Methods. The average length ± the standard error is plotted above for each time point for 100 measured spirochetes.

FIG 5

Determination of the relative DNA content of wild-type (GCB908) versus resT-inducible spirochetes (GCB2127) by quantification of fluorescence intensity of Hoechst 33258-stained spirochetes in fluorescence micrographs. Images of fluorescent spirochetes were analyzed for total fluorescence intensity by collecting the integrated fluorescence density using ImageJ (NIH) and correcting for the fluorescence background. The mean fluorescence intensity ± the standard error is plotted. Statistical significance was determined by a two-tailed unpaired t test and is indicated above the graphs (*, P < 0.05; **, P < 0.01; n.s., not significant).

FIG 6

Field inversion gel of isolated B. burgdorferi genomic DNA. The resT-inducible strain GCB2127 (lanes 1 and 2) was grown for 48 h with or without IPTG, and the genomic content was compared to that of the wild type, B31-A (GCB908) (lanes 3 and 4). The gel was stained with ethidium bromide. M, lambda monocut size marker (NEB).

FIG 7

Southern blot of genomic DNA from a resT-inducible strain (GCB2127). Spirochetes were grown either with or without IPTG, and the culture was sampled at 0, 24, 48, 72, and 96 h. DNA was extracted and run on a 0.65% agarose field inversion gel and transferred to a nylon membrane for Southern blotting. Probes were generated against the left end of lp17 (A) as well as the right end of lp28-2 (B) as described in Materials and Methods, using PCR primers B2275 and B2276 for lp17 and B2295 and B2296 for lp28-2.

FIG 8

Treatment of DNA from the resT-inducible strain (GCB2127) and the wild-type parent (GCB908) with the telomere resolvase ResT. (A) Schematic of the experimental design. To simplify detection of telomere ends, DNA was digested with a restriction enzyme to liberate either a free telomere or the replicated telomere junction. ResT activity on the replicated junction generates free ends, which were detected by Southern blotting hybridization. (B and C) Southern blots of DNA that was isolated 72 h after IPTG washout and digested with EcoRI or BamHI prior to being treated with ResT. DNA was run on a 1% agarose gel and transferred to a nylon membrane for Southern blotting hybridization with probes for the lp17 (B) and lp28-2 (C) left ends.